Efrain Sanchez-Ortiz

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus–9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

TrkA Gene Ablation in Basal Forebrain Results in Dysfunction of the Cholinergic Circuitry

Dysfunction of basal forebrain cholinergic neurons (BFCNs) is an early pathological hallmark of Alzheimers disease (AD). Numerous studies have indicated that nerve growth factor (NGF) supports survival and phenotypic differentiation of BFCNs. Consistent with a potential link to AD pathogenesis, TrkA, a NGF receptor, is expressed in cholinergic forebrain neuronal populations including those in BF and striatum, and is markedly reduced in individuals with mild cognitive impairment (MCI) without dementia and early-stage AD. To investigate the role of TrkA in the development, connectivity, and function of the BF cholinergic system and its contribution to AD pathology, we have generated a forebrain-specific conditional TrkA knock-out mouse line. Our findings show a key role for TrkA signaling in establishing the BF cholinergic circuitry through the ERK pathway, and demonstrate that the normal developmental increase of choline acetyltransferase expression becomes critically dependent on TrkA signaling before neuronal connections are established. Moreover, the anatomical and physiological deficits caused by lack of TrkA signaling in BFCNs have selective impact on cognitive activity. These data demonstrate that TrkA loss results in cholinergic BF dysfunction and cognitive decline that is reminiscent of MCI and early AD.

Control of muscle formation by the fusogenic micropeptide myomixer

Micromanaging muscle cell fusion Adult skeletal muscles are characterized by long, multinucleated cells called myofibers. Myofibers form when muscle precursor cells, or myoblasts, differentiate and fuse together during embryogenesis. The fusion process is not fully understood. Studying cell culture and mouse models, Bi et al. identified an 84–amino acid peptide that promotes myoblast fusion. This small peptide, called Myomixer, physically interacts with and stimulates the activity of a fusogenic membrane protein called Myomaker. Notably, the Myomaker-Myomixer pair can also promote the fusion of nonmuscle cells, such as fibroblasts. Science, this issue p. 323 A small peptide expressed in developing skeletal muscle controls muscle cell fusion and myofiber formation. Skeletal muscle formation occurs through fusion of myoblasts to form multinucleated myofibers. From a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function screen for genes required for myoblast fusion and myogenesis, we discovered an 84–amino acid muscle-specific peptide that we call Myomixer. Myomixer expression coincides with myoblast differentiation and is essential for fusion and skeletal muscle formation during embryogenesis. Myomixer localizes to the plasma membrane, where it promotes myoblast fusion and associates with Myomaker, a fusogenic membrane protein. Myomixer together with Myomaker can also induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude that the Myomixer-Myomaker pair controls the critical step in myofiber formation during muscle development.

NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development

Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders.

Analysis of FMR1 deletion in a subpopulation of post-mitotic neurons in mouse cortex and hippocampus.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the leading cause of autism. FXS is caused by mutation in a single gene, FMR1, which encodes an RNA‐binding protein FMRP. FMRP is highly expressed in neurons and is hypothesized to have a role in synaptic structure, function, and plasticity by regulating mRNAs that encode pre‐ and post‐synaptic proteins. Fmr1 knockout (KO) mice have been used as a model to study FXS. These mice have been reported to show a great degree of phenotypic variability based on the genetic background, environmental signals, and experimental methods. In this study, we sought to restrict FMRP deletion to two brain regions that have been implicated in FXS and autism. We show that ablating Fmr1 in differentiated neurons of hippocampus and cortex results in dendritic alterations and changes in synaptic marker intensity that are brain region specific. In our conditional mutant mice, FMRP‐deleted neurons have activated AKT‐mTOR pathway signaling in hippocampus but display no apparent behavioral phenotypes. These results highlight the importance of identifying additional factors that interact with Fmr1 to develop FXS. Autism Res 2014, 7: 60–71.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Single-cut correction of a dystrophin gene mutation with CRISPR/Cas9 restored dystrophin expression in skeletal and cardiac muscles in a mouse model of Duchenne muscular dystrophy. Making the cut Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a fatal childhood muscle disease. To optimize the correction of DMD mutations by CRISPR/Cas9 gene editing, Amoasii et al. first generated mice that had exon 50 deleted, a common human mutational “hotspot” region of the dystrophin gene. The authors then reported a method in which a single cut in genomic DNA encoding dystrophin with CRISPR/Cas9 in these engineered mice resulted in up to 90% restoration of dystrophin expression in mouse skeletal and heart muscles. This method of permanently bypassing DMD mutations using a single genomic cut suggests that this strategy may have potential for efficiently correcting DMD mutations. Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration

Significance Skeletal muscle damaged by injury or disease can regenerate new muscle fibers. The regenerative properties of skeletal muscle involve fusion of activated muscle stem cells (satellite cells). We recently discovered Myomixer, a conserved micropeptide that is specifically expressed during muscle formation. Myomixer, together with its partner Myomaker, another muscle-specific membrane protein, is necessary for muscle formation during embryogenesis. Here, we show the absolute requirement of Myomixer for the fusion of satellite cells and regeneration of adult muscle in response to injury. Our findings provide insights into the mechanisms of muscle formation and suggest opportunities for enhancing muscle regeneration through manipulation of Myomixer and Myomaker. Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Gene editing and muscular dystrophy Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and a shortened life span. The disease is caused by mutations that reduce or prevent expression of dystrophin, an essential structural protein in skeletal and heart muscle. The gene editing technology CRISPR-Cas9 can correct disease-causing mutations and has yielded promising results in mouse models of DMD. In a small, short-term study, Amoasii et al. tested this strategy in a dog model of DMD that exhibits many features of the human disease. Intramuscular or systemic delivery of the gene editing components resulted in a substantial increase in dystrophin protein levels in skeletal and heart muscle. Restoration of dystrophin expression was accompanied by improved muscle histology. Science, this issue p. 86 Successful CRISPR correction of a dystrophin mutation in dogs increases dystrophin protein expression in skeletal and heart muscle. Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

Identification of a multipotent Twist2-expressing cell population in the adult heart

Significance Adult mammalian hearts have limited self-renewal capacity. Although mounting evidence indicates that new cardiomyocytes are derived from dedifferentiation and proliferation of existing cardiomyocytes, the contribution of adult cardiac progenitors to cardiomyocyte renewal during homeostasis and upon injury remains under debate. The basic helix–loop–helix transcription factor Twist2 is expressed in interstitial cells in the adult myocardium. Using genetic lineage tracing, we identified a Twist2-expressing cell population that gives rise to a small number of adult cardiomyocytes in vivo. These Twist2-expressing cells can differentiate into cardiomyocytes, endothelial cells, and fibroblasts in culture and contribute to cardiac renewal through cell fusion and de novo differentiation. Our findings add Twist2-expressing cells to the cellular constituents involved in adult cardiac maintenance and remodeling. Twist transcription factors function as ancestral regulators of mesodermal cell fates in organisms ranging from Drosophila to mammals. Through lineage tracing of Twist2 (Tw2)-expressing cells with tamoxifen-inducible Tw2-CreERT2 and tdTomato (tdTO) reporter mice, we discovered a unique cell population that progressively contributes to cardiomyocytes (CMs), endothelial cells, and fibroblasts in the adult heart. Clonal analysis confirmed the ability of Tw2-derived tdTO+ (Tw2-tdTO+) cells to form CMs in vitro. Within the adult heart, Tw2-tdTO+ CMs accounted for ∼13% of total CMs, the majority of which resulted from fusion of Tw2-tdTO+ cells with existing CMs. Tw2-tdTO+ cells also contribute to cardiac remodeling after injury. We conclude that Tw2-tdTO+ cells participate in lifelong maintenance of cardiac function, at least in part through de novo formation of CMs and fusion with preexisting CMs, as well as in the genesis of other cellular components of the adult heart.